1. Field of the Invention
The present invention relates to the field of viral proteins, particularly those proteins involved in HIV gene regulation and mutants thereof. The gene which encodes the viral factor is also related to the field of the present invention. Recombinant vectors and host cells including the gene for the viral nucleic acid binding factor is also related to the present disclosure. The present invention also relates to the field of methods for regulating the expression of cellular and viral genes, particularly HIV gene expression, and to methods of treatment, and therapeutic agents for treating acquired immunodeficiency disease and other HIV related diseases or symptoms incident an HIV or AIDS infection.
2. Background of the Related Art
The regulation of HIV-1 gene expression is dependent on a number of cis-acting elements located in the LTR. Both DNA and RNA elements in the HIV-1 LTR serve as binding sites for cellular factors. In addition, viral regulatory proteins such as Tat and rev are involved in the activation of gene HIV-1 expression. The mechanisms by which cellular factors interact with Tat and rev to increase HIV-1 gene expression are not understood.
The human immunodeficiency virus (HIV) is the causative agent of AIDS..sup.2,26 In common with other retroviruses.sup.10, HIV contains two long terminal repeats (LTRs) and three conserved genes, gag, pol, and env. It also contains a number of critical regulatory genes including tat and rev which, in conjunction with cellular polymerases and transcription factors, are necessary for the activation of viral gene expression..sup.7,14,21 Once HIV-1 integrates into the host cell genome, its gene expression is regulated by cellular transcription factors in a manner similar to that of endogenous cellular genes..sup.10 Unlike cellular genes, unique features in the DNA and RNA regulatory regions of the HIV-1 LTR make it a target for the viral transactivator protein, Tat. The activities of several of the cellular transcription factors which bind to the HIV-1 LTR are altered by parameters such as activation or differentiation of lymphocytes or macrophages, the action of lymphokines, and alterations of signal transduction pathways..sup.12,35 Thus, HIV is subject to many of the same regulatory signals that are important in controlling cellular gene expression.
A variety of viral transactivators including the adenovirus E1A, cytomegalovirus immediate early, and the human T-cell leukemia virus tax proteins are able to activate HIV-1 gene expression..sup.6,32 These proteins activate HIV-1 through different regulatory elements including the TATA box, USF binding site, and NF-kB sites respectively..sup.6,32 In contrast to these viral transactivator proteins whose activity is relatively permissive, activation by the Tat protein is specific for HIV. Disruption of the tat gene prevents viral replication indicating its essential role in the HIV-1 life cycle..sup.14,21 The Tat protein is known to possess at least three functional domains..sup.28 These include an amino-terminal activation domain, a cysteine-rich domain which may function in dimerization, and a basic domain which is important in nuclear localization and RNA binding..sup.8,9,11,15,16,28 Despite knowledge of these details, the mechanism of Tat activation remains open to question, and thus an identification of potential interactions between Tat and cellular factors is important for understanding Tat function.
A number of cis-acting regulatory elements in the HIV-1 LTR are critical for basal and Tat-induced gene expression. These include the enhancer, SP1.sup.38,45, TATA28.sup.29,46 and TAR elements.sup.3,19,29,44. Each of these elements serves as a binding site for cellular transcription factors. Though the SP1 and TATA elements influence the basal level of HIV-1 gene expression, they also play a role in determining the level of activation by the transactivator protein Tat..sup.45 The TAR element which forms a stable stem-loop RNA structure extending from +1 to +60 is critical for Tat activation..sup.3,19,24,44 A number of studies using fusions of Tat to other known DNA or RNA binding proteins indicate that Tat is able to activate HIV-1 gene expression when bound to either DNA or RNA..sup.41,45 Thus it is likely that factors binding to both DNA and RNA regulatory elements influence the degree of Tat activation.
Mutagenesis has localized a region of TAR RNA between +18 and +44 as an essential element for activation by Tat..sup.3-5,13,19,29,42 Several elements in this RNA region including the bulge (+23/+25), the loop (+30/+35), and the stem structure are required for complete Tat-activation..sup.3,5,13,19,29,42 The function of the stem structure is likely to maintain the position of the bulge and loop structures. The bulge region in TAR RNA serves as the binding site for Tat though the loop sequences also influence Tat binding..sup.8,9,11,16 In addition, cellular factors are also capable of binding to the bulge sequences. The interaction between Tat and the TAR RNA bulge is very specific in that a change of one nucleotide at +23 in the bulge is sufficient to disrupt Tat binding..sup.8,9,11,16 The basic domain of Tat is necessary and sufficient for binding to the TAR RNA bulge..sup.8,9,11,16 Extensive mutagenesis of the Tat protein indicates that arginine residues at positions 52 and 53 of Tat are especially critical for interacting with phosphate groups in the TAR RNA bulge..sup.9 The Tat binding to the TAR RNA bulge is thus highly specific and of great affinity.
In contrast to the bulge which binds a viral protein, the loop sequences serve as a binding site for cellular factors that may cooperate with Tat in activating HIV-1 gene expression..sup.30,31 Fractionation of HeLa nuclear extract and gel retardation and UV crosslinking using TAR RNA probes indicate that two different cellular proteins p68 and TRP-185 bind to the TAR RNA loop sequences. TRP-185 is a ubiquitously expressed 185kDa protein whose binding to TAR RNA is regulated by additional cofactor proteins. These cofactors likely function by post-translational modification of TRP-185 i.e., phosphorylation. TRP-185 binding to TAR RNA requires wild-type loop sequences and an intact bulge structure. The binding of TRP-185 to TAR RNA, unlike that of Tat, is not markedly influenced by the primary sequences of the bulge region. Both Tat and TRP-185 activate HIV-1 LTR gene expression in in vitro transcription assays, but whether these proteins directly interact is not known. These results indicate that Tat activation via the TAR element may require interactions between Tat and cellular transcription factors.
Activation of the HIV-1 LTR by Tat proteins with an altered basic domain has previously been demonstrated to be strongly dependent on the concentration of transfected DNA (Ruben et al., 1989; Hauber et al., 1989). However, how this finding relates to the overall activation of the HIV-1 LTR remains to be determined.
It is critical to determine how Tat modulates the transcriptional apparatus to increase HIV-1 gene expression. Tat stimulates steady state RNA levels synthesized from the HIV-1 LTR approximately 20 to 50-fold. Nuclear run-on experiments using the HIV-1 LTR indicate that Tat stimulates transcriptional initiation. However another effect of Tat function is seen when nascent RNA is measured at various positions downstream of the HIV-1 LTR initiation site in both the presence and absence of Tat..sup.18 Though several studies demonstrate an increased number of RNA molecules synthesized from proximal portions of the HIV-1 LTR (near the initiation site) in the presence of Tat, the predominant effect of Tat appears to be a marked increase in the level of RNA synthesized at promoter distal sites (between 500 to 1000 nucleotides from the initiation site)..sup.18 In vitro analysis of Tat transactivation also supports an effect on transcriptional elongation. The ability of Tat to increase the number of elongated transcripts may be one explanation for the decrease in the number of short transcripts which are synthesized from the HIV-1 LTR in the absence of Tat. These short transcripts terminate around +60 in the TAR element and may reflect the products of poorly processive transcription complexes. Thus, Tat may function at multiple steps in the transcriptional pathway to increase both the initiation and elongation of transcripts from the HIV LTR.
Mutations in a number of HIV-1 genes including tat (Pearson et al. (1990)), rev ((Malin et al. (1989)), and gag (Trono et al. (1989)) result in proteins with a dominant negative or transdominant phenotype that interfere with the function of the corresponding wild-type proteins. Recently, a .DELTA.tat mutation has been described by the present inventors. The .DELTA.tat mutant gene therein encoded a 54 amino acid length HIV protein having truncated basic domain (Pearson et al. (1990)). The "basic domain" of the tat gene includes 9 amino acids and is defined by amino acid residues 49-57 of the first 72 amino acids encoding the Tat protein (Pearson et al. (1990)). Three (3) of the amino acid residues of the basic domain of the HIV Tat protein were eliminated in the .DELTA.tat to provide the final protein product, leaving six (6) of the residues of the basic domain unchanged. While the .DELTA.tat-encoded protein was found to inhibit Tat activation of the HIV-1 LTR when the vector expressing it was present in a 5- to 30-fold molar excess over a vector expressing the wild-type Tat, the mutations were not found to result in a transdominant phenotype.
Further characterization of the precise mechanisms controlling HIV gene expression in regard to the role of the "basic domain" of the tat gene has not been explored, despite the impact such would have in providing more potent and effective therapeutic agents for treating HIV infections.
Previous data have demonstrated that Tat protein is capable of entering cells in culture when added to the tissue culture media..sup.23 Though the mechanism of entry is not understood it appears to be a result of endocytosis. To develop transdominant Tat mutant peptides for potential therapeutic use it would be important to develop transdominant mutants of minimal size. This is due to the fact that the amount of partial products and the yield of peptides decrease significantly as their size is increased. A construct which encoded a peptide capable of providing defective activation of HIV LTR gene expression and an ability to antagonize wild-type Tat function, and which was of sufficiently small size to optimize partial product and peptide yield would enable the production of an entirely new class of therapeutic agents used in the treatment and potential cure of HIV infections.